Anti-pd-1 antibodies

ABSTRACT

Anti-PD-1 antibodies are disclosed. Also disclosed are pharmaceutical compositions comprising such antibodies, and uses and methods using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of Internationalapplication serial no. PCT/SG2015/050413 (WO 2016/068801), filed Oct.27, 2015, entitled “Anti-PD-1 Antibodies”. International applicationserial no. PCT/SG2015/050413 claims priority to GB application serialno. 1419084.7, filed Oct. 27, 2014. All of the referenced applicationsare incorporated herein by reference.

REFERENCE TO A “SEQUENCE LISTING”

Incorporated by reference herein in its entirety is the Sequence Listingentitled “sequence Listing.txt”, created Apr. 21, 2017, size of 61kilobytes.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to programmed celldeath 1 (PD-1).

BACKGROUND TO THE INVENTION

T-cell exhaustion is a state of T-cell dysfunction that arises duringmany chronic infections and cancer. It is defined by poor T-celleffector function, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T-cells. Exhaustion prevents optimal control of infection andtumors. (E John Wherry., Nature Immunology 12, 492-499 (2011)).

T-cell exhaustion is characterized by the stepwise and progressive lossof T-cell functions. Exhaustion is well-defined during chroniclymphocytic choriomeningitis virus infection and commonly develops underconditions of antigen-persistence, which occur following many chronicinfections including hepatitis B virus, hepatitis C virus and humanimmunodeficiency virus infections, as well as during tumor metastasis.Exhaustion is not a uniformly disabled setting as a gradation ofphenotypic and functional defects can manifest, and these cells aredistinct from prototypic effector, memory and also anergic T cells.Exhausted T cells most commonly emerge during high-grade chronicinfections, and the levels and duration of antigenic stimulation arecritical determinants of the process. (Yi et al., Immunology April 2010;129(4):474-481).

Circulating human tumor-specific CD8⁺ T cells may be cytotoxic andproduce cytokines in vivo, indicating that self- and tumor-specifichuman CD8⁺ T cells can reach functional competence after potentimmunotherapy such as vaccination with peptide, incomplete Freund'sadjuvant (IFA), and CpG or after adoptive transfer. In contrast toperipheral blood, T-cells from metastasis are functionally deficient,with abnormally low cytokine production and upregulation of theinhibitory receptors PD-1, CTLA-4, and TIM-3. Functional deficiency isreversible, since T-cells isolated from melanoma tissue can restoreIFN-γ production after short-term in vitro culture. However, it remainsto be determined whether this functional impairment involves furthermolecular pathways, possibly resembling T-cell exhaustion or anergy asdefined in animal models. (Baitsch et al., J Clin Invest. 2011;121(6):2350-2360).

Programmed cell death 1 (PD-1), also called CD279, is a type I membraneprotein encoded in humans by the PDCD1 gene. It has two ligands, PD-L1and PD-L2.

The PD-1 pathway is a key immune-inhibitory mediator of T-cellexhaustion. Blockade of this pathway can lead to T-cell activation,expansion, and enhanced effector functions. As such, PD-1 negativelyregulates T cell responses. PD-1 has been identified as a marker ofexhausted T cells in chronic disease states, and blockade of PD-1; PD-1Linteractions has been shown to partially restore T cell function.(Sakuishi et ale, JEM Vol. 207, Sep. 27, 2010, pp 2187-2194).

Nivolumab (BMS-936558) is an anti-PD-1 antibody that was approved forthe treatment of melanoma in Japan in July 2014. Other anti-PD-1antibodies are described in WO 2010/077634, WO 2006/121168,WO2008/156712 and WO2012/135408.

T cell immunoglobulin mucin 3 (TIM-3) is an immune regulator identifiedas being upregulated on exhausted CD8⁺ T cells (Sakuishi et al., JEMVol. 207, Sep. 27, 2010, pp 2187-2194). TIM-3 was originally identifiedas being selectively expressed on IFN-γ-secreting Th1 and Tc1 cells.Interaction of TIM-3 with its ligand, galectin-9, triggers cell death inTIM-3⁺ T cells. Anti-TIM-3 antibodies are described in Ngiow et al(Cancer Res. 2011 May 15; 71(10):3540-51), and in U.S. Pat. No.8,552,156

Both TIM-3 and PD-1 can function as negative regulators of T cellresponses and combined targeting of the TIM-3 and PD-1 pathways is moreeffective in controlling tumor growth than targeting either pathwayalone. (Sakuishi et al., JEM Vol. 207, Sep. 27, 2010, pp 2187-2194; andNgiow et al Cancer Res. 2011 May 15; 71(10):3540-51).

SUMMARY OF THE INVENTION

The present invention is concerned with antibodies, or antigen bindingfragments, that bind to PD-1. Heavy and light chain polypeptides arealso disclosed. The antibodies, antigen binding fragments andpolypeptides may be provided in isolated and/or purified form and may beformulated into compositions suitable for use in research, therapy anddiagnosis.

In some embodiments the antibody, or antigen binding fragment, orpolypeptide may be effective to restore T-cell function in T-cells, e.g.CD8⁺ T-cells, exhibiting T-cell exhaustion or T-cell anergy.

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the amino acid sequence of the antibody maycomprise the amino acid sequences i) to iii), or the amino acidsequences iv) to vi), or preferably the amino acid sequences i) to vi):

i) LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN ii) LC-CDR2: (SEQ ID NO: 26)SNNQRPS iii) LC-CDR3: (SEQ ID NO: 53) X₁X₂WDDX₃X₄X₅GX₆X₇ iv) HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or HC-CDR1: (SEQ ID NO: 89) SYGMH v) HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG vi) HC-CDR3: (SEQ ID NO: 54)DZ₁GZ₂GZ₃YZ₄YGZ₅DZ₆or a variant thereof in which one or two or three amino acids in one ormore of the sequences (i) to (vi) are replaced with another amino acid,where X₁=A or S, X₂=S or A, X₃=V, Y, F, D, S or A, X₄=L, Y, V or A,X₅=Y, R or H, X₆=S, or T, X₇=V, I, or M and Z₁=L or Y, Z₂=A or S, Z₃=Por Y, Z₄=Y or L, Z₅=K, M or L, Z₆=H or V.

In connection with all aspects of the present invention, in embodimentswherein HC-CDR1: SYGMH (SEQ ID NO:89), this sequence may be comprised inthe larger sequence GFTFSSYGMH (SEQ ID NO:39).

In some embodiments, LC-CDR3 is one of ASWDDVLYGSV (SEQ ID NO:27),ASWDDYYYGTI (SEQ ID NO:28), ASWDDYLRGTV (SEQ ID NO:29), SAWDDYLHGTV (SEQID NO:30), ASWDDYVRGTM (SEQ ID NO:31), SSWDDFLRGTV (SEQ ID NO:32),SSWDDDARGTI (SEQ ID NO:33), AAWDDVYYGTI (SEQ ID NO:34), ASWDDSLYGTV (SEQID NO:35), AAWDDAYYGTI (SEQ ID NO:36), ASWDDVYRGTV (SEQ ID NO:37), orSSWDDSLYGTI (SEQ ID NO:38). In some embodiments HC-CDR3 is one ofDLGAGPYYYGKDH (SEQ ID NO:41), DLGAGPYYYGKDV (SEQ ID NO:42),DYGAGPYYYGMDV (SEQ ID NO:43), DLGAGPYYYGLDV (SEQ ID NO:44),DLGAGPYYYGMDV (SEQ ID NO:45), DLGAGPYYYGMDV (SEQ ID NO:46),DLGAGPYYYGMDV (SEQ ID NO:47), DLGAGPYYYGMDV (SEQ ID NO:48),DLGAGPYYYGMDV (SEQ ID NO:49), DYGAGPYYYGMDV (SEQ ID NO:50),DLGSGYYLYGMDV (SEQ ID NO:51), or DLGAGPYYYGMDV (SEQ ID NO:52).

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 27) ASWDDVLYGSV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 28) ASWDDYYYGTI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 29) ASWDDYLRGTV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 30) SAWDDYLHGTV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 31) ASWDDYVRGTM

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 32) SSWDDFLRGTV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 33) SSWDDDARGTI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 34) AAWDDVYYGTI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 35) ASWDDSLYGTV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 36) AAWDDAYYGTI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 37) ASWDDVYRGTV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 38) SSWDDSLYGTI

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 41) DLGAGPYYYGKDH

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 42) DLGAGPYYYGKDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 43) DYGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 44) DLGAGPYYYGLDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 45) DLGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 46) DLGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 47) DLGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 48) DLGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 49) DLGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 50) DYGAGPYYYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 51) DLGSGYYLYGMDV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 52) DLGAGPYYYGMDV

The antibody may comprise at least one light chain variable regionincorporating the CDRs shown in FIG. 1A-F or 3A-C. The antibody maycomprise at least one heavy chain variable region incorporating the CDRsshown in FIG. 2A-F or 3A-C.

The antibody may comprise at least one light chain variable region(V_(L)) comprising the amino acid sequence of one of SEQ ID NOs 1, 25,26, 27 or 2, 25, 26, 28, or 3, 25, 26, 29 or 4, 25, 26, 30, or 5, 25,26, 31 or 6, 25, 26, 32 or 7, 25, 26, 33 or 8, 25, 26, 34 or 9, 25, 26,35 or 10, 25, 26 36 or 11, 25, 26, 37 or 12, 25, 26, 38, or one of theamino acid sequences shown in FIG. 1A-F or an amino acid sequence havingat least 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%,sequence identity to one of SEQ ID NOs 1, 25, 26, 27 or 2, 25, 26, 28,or 3, 25, 26, 29 or 4, 25, 26, 30, or 5, 25, 26, 31 or 6, 25, 26, 32 or7, 25, 26, 33 or 8, 25, 26, 34 or 9, 25, 26, 35 or 10, 25, 26 36 or 11,25, 26, 37 or 12, 25, 26, 38, or to the amino acid sequence of the V_(L)chain amino acid sequence shown in FIG. 1A-F.

The antibody may comprise at least one heavy chain variable region(V_(H)) comprising the amino acid sequence of one of SEQ ID NOs 13, 39or 89, 40, 41 or 14, 39 or 89, 40, 42, or 15, 39 or 89, 40, 43 or 16, 39or 89, 40, 44 or 17, 39 or 89, 40, 45 or 18, 39 or 89, 40, 46 or 19, 39or 89, 40, 47 or 20, 39 or 89, 40, 48 or 21, 39 or 89, 40, 49 or 22, 39or 89, 40, 50 or 23, 39 or 89, 40, 51 or 24, 39 or 89, 40, 52, or one ofthe amino acid sequences shown in FIG. 2A-F or an amino acid sequencehaving at least 70%, more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to one of SEQ ID NOs 13, 39, 40, 41 or 14, 39 or89, 40, 42, or 15, 39 or 89, 40, 43 or 16, 39 or 89, 40, 44 or 17, 39 or89, 40, 45 or 18, 39 or 89, 40, 46 or 19, 39 or 89, 40, 47 or 20, 39 or89, 40, 48 or 21, 39 or 89, 40, 49 or 22, 39 or 89, 40, 50 or 23, 39 or89, 40, 51 or 24, 39 or 89, 40, 52, or to the amino acid sequence of theV_(H) chain amino acid sequence shown in FIG. 2A-F.

The antibody may comprise at least one light chain variable regioncomprising the amino acid sequence of one of SEQ ID NOs 1, 25, 26, 27 or2, 25, 26, 28, or 3, 25, 26, 29 or 4, 25, 26, 30, or 5, 25, 26, 31 or 6,25, 26, 32 or 7, 25, 26, 33 or 8, 25, 26, 34 or 9, 25, 26, 35 or 10, 25,26 36 or 11, 25, 26, 37 or 12, 25, 26, 38, or to one of the amino acidsequences shown in FIG. 1A-F (or an amino acid sequence having at least70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100%, sequence identity to one of SEQ ID NOs 1, 25, 26, 27or 2, 25, 26, 28, or 3, 25, 26, 29 or 4, 25, 26, 30, or 5, 25, 26, 31 or6, 25, 26, 32 or 7, 25, 26, 33 or 8, 25, 26, 34 or 9, 25, 26, 35 or 10,25, 26 36 or 11, 25, 26, 37 or 12, 25, 26, 38, or to one of the aminoacid sequences of the V_(L) chain amino acid sequence shown in FIG.1A-F) and at least one heavy chain variable region comprising the aminoacid sequence of one of SEQ ID NOs 13, 39 or 89, 40, 41 or 14, 39 or 89,40, 42, or 15, 39 or 89, 40, 43 or 16, 39 or 89, 40, 44 or 17, 39 or 89,40, 45 or 18, 39 or 89, 40, 46 or 19, 39 or 89, 40, 47 or 20, 39 or 89,40, 48 or 21, 39 or 89, 40, 49 or 22, 39 or 89, 40, 50 or 23, 39 or 89,40, 51 or 24, 39 or 89, 40, 52, or one of the amino acid sequence shownin FIG. 2A-F (or an amino acid sequence having at least 70%, morepreferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity toone of SEQ ID NOs 13, 39 or 89, 40, 41 or 14, 39 or 89, 40, 42, or 15,39 or 89, 40, 43 or 16, 39 or 89, 40, 44 or 17, 39 or 89, 40, 45 or 18,39 or 89, 40, 46 or 19, 39 or 89, 40, 47 or 20, 39 or 89, 40, 48 or 21,39 or 89, 40, 49 or 22, 39 or 89, 40, 50 or 23, 39 or 89, 40, 51 or 24,39 or 89, 40, 52, or to one of the amino acid sequences of the V_(H)chain amino acid sequence shown in FIG. 2A-F).

The antibody may optionally bind PD-1. The antibody may optionally haveamino acid sequence components as described above. The antibody may bean IgG. In one embodiment an in vitro complex, optionally isolated,comprising an antibody, or antigen binding fragment, as describedherein, bound to PD-1 is provided.

In one aspect of the present invention an isolated heavy chain variableregion polypeptide is provided, the heavy chain variable regionpolypeptide comprising the following CDRs:

HC-CDR1: (SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2:(SEQ ID NO: 40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 54)DZ₁GZ₂GZ₃YZ₄YGZ₅DZ₆where Z₁=L or Y, Z₂=A or S, Z₃=P or Y, Z₄=Y or L, Z₅=K, M or L, Z₆=H orV.

In some embodiments HC-CDR3 is one of DLGAGPYYYGKDH (SEQ ID NO:41),DLGAGPYYYGKDV (SEQ ID NO:42), DYGAGPYYYGMDV (SEQ ID NO:43),DLGAGPYYYGLDV (SEQ ID NO:44), DLGAGPYYYGMDV (SEQ ID NO:45),DLGAGPYYYGMDV (SEQ ID NO:46), DLGAGPYYYGMDV (SEQ ID NO:47),DLGAGPYYYGMDV (SEQ ID NO:48), DLGAGPYYYGMDV (SEQ ID NO:49),DYGAGPYYYGMDV (SEQ ID NO:50), DLGSGYYLYGMDV (SEQ ID NO:51), orDLGAGPYYYGMDV (SEQ ID NO:52).

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the antibody, or antigen binding fragment,comprising a heavy chain and a light chain variable region sequence,wherein:

-   -   the heavy chain comprises a HC-CDR1, HC-CDR2, HC-CDR3, having at        least 85% overall sequence identity to    -   HC-CDR1: GFTFSSYGMH (SEQ ID NO:39) or SYGMH (SEQ ID NO:89),    -   HC-CDR2 VISYDGSNKYYADSVKG (SEQ ID NO:40),    -   HC-CDR3: is one of DZ₁GZ₂GZ₃YZ₄YGZ₅DZ₆ (SEQ ID NO:54),        DLGAGPYYYGKDH (SEQ ID NO:41), DLGAGPYYYGKDV (SEQ ID NO:42),        DYGAGPYYYGMDV (SEQ ID NO:43), DLGAGPYYYGLDV (SEQ ID NO:44),        DLGAGPYYYGMDV (SEQ ID NO:45), DLGAGPYYYGMDV (SEQ ID NO:46),        DLGAGPYYYGMDV (SEQ ID NO:47), DLGAGPYYYGMDV (SEQ ID NO:48),        DLGAGPYYYGMDV (SEQ ID NO:49), DYGAGPYYYGMDV (SEQ ID NO:50),        DLGSGYYLYGMDV (SEQ ID NO:51), or DLGAGPYYYGMDV (SEQ ID NO:52)        respectively, where Z₁=L or Y, Z₂=A or S, Z₃=P or Y, Z₄=Y or L,        Z₅=K, M or L, Z₆=H or V, and    -   the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at        least 85% overall sequence identity to    -   LC-CDR1: SGSSSNIKFNSVN (SEQ ID NO:25),    -   LC-CDR2: SNNQRPS (SEQ ID NO:26),    -   LC-CDR3: is one of X₁X₂WDDX₃X₄X₅GX₆X₇(SEQ ID NO:53), ASWDDVLYGSV        (SEQ ID NO:27), ASWDDYYYGTI (SEQ ID NO:28), ASWDDYLRGTV (SEQ ID        NO:29), SAWDDYLHGTV (SEQ ID NO:30), ASWDDYVRGTM (SEQ ID NO:31),        SSWDDFLRGTV (SEQ ID NO:32), SSWDDDARGTI (SEQ ID NO:33),        AAWDDVYYGTI (SEQ ID NO:34), ASWDDSLYGTV (SEQ ID NO:35),        AAWDDAYYGTI (SEQ ID NO:36), ASWDDVYRGTV (SEQ ID NO:37), or        SSWDDSLYGTI (SEQ ID NO:38) respectively, where X₁=A or S, X₂=S        or A, X₃=V, Y, F, D, S or A, X₄=L, Y, V or A, X₅=Y, R or H,        X₆=S, or T, X₇=V, I, or M.

In some embodiments the degree of sequence identity may be one of 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In another aspect of the present invention an antibody, or antigenbinding fragment, optionally isolated, is provided comprising a heavychain and a light chain variable region sequence, wherein:

-   -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of one of SEQ ID NOs:13 to 24 (FIG.        2A-F), and    -   the light chain sequence has at least 85% sequence identity to        the light chain sequence of one of: SEQ ID NOs:1 to 12 (FIG.        1A-F).

In some embodiments the degree of sequence identity may be one of 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region heavy chain frameworksequences between the CDRs according to the arrangementHCFR1:HC-CDR1:HCFR2:HC-CDR2:HCFR3:HC-CDR3:HCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In one aspect of the present invention an isolated light chain variableregion polypeptide, optionally in combination with a heavy chainvariable region polypeptide as described herein, is provided, the lightchain variable region polypeptide comprising the following CDRs:

LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 53) X₁X₂WDDX₃X₄X₅GX₆X₇where X₁=A or S, X₂=S or A, X₃=V, Y, F, D, S or A, X₄=L, Y, V or A,X₅=Y, R or H, X₆=S, or T, X₇=V, I, or M.

In some embodiments LC-CDR3 is one of ASWDDVLYGSV (SEQ ID NO:27),ASWDDYYYGTI (SEQ ID NO:28), ASWDDYLRGTV (SEQ ID NO:29), SAWDDYLHGTV (SEQID NO:30), ASWDDYVRGTM (SEQ ID NO:31), SSWDDFLRGTV (SEQ ID NO:32),SSWDDDARGTI (SEQ ID NO:33), AAWDDVYYGTI (SEQ ID NO:34), ASWDDSLYGTV (SEQID NO:35), AAWDDAYYGTI (SEQ ID NO:36), ASWDDVYRGTV (SEQ ID NO:37), orSSWDDSLYGTI (SEQ ID NO:38).

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region light chain frameworksequences between the CDRs according to the arrangementLCFR1:LC-CDR1:LCFR2:LC-CDR2:LCFR3:LC-CDR3:LCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a human constant region. For example selected from oneof IgG1, IgG2, IgG3 and IgG4.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a murine constant region. For example, selected fromone of IgG1, IgG2A, IgG2B and IgG3.

In another aspect of the present invention, an antibody or antigenbinding fragment is provided, optionally isolated, which is capable ofbinding to PD-1, and which is a bispecific antibody or a bispecificantigen binding fragment. In some embodiments, the bispecific antibodyor bispecific antigen binding fragment comprises an antigen bindingfragment or polypeptide capable of binding to PD-1 as described herein,and additionally comprises an antigen binding domain which is capable ofbinding to another target protein, e.g. a target protein other thanPD-1. In some embodiments, the target protein is a cell surfacereceptor. In some embodiments, the target protein is a cell surfacereceptor expressed on the cell surface of immune cells, e.g. T cells. Insome embodiments, the target protein may be a member of the CD28 family.In some embodiments, the member of the CD28 family is selected fromTIM-3, LAGS, ICOS, CTLA4, BTLA or CD28.

In another aspect of the present invention, a composition, e.g. apharmaceutical composition or medicament, is provided. The compositionmay comprise an antibody, antigen binding fragment, or polypeptide asdescribed herein and at least one pharmaceutically-acceptable carrier,excipient, adjuvant or diluent.

In another aspect of the present invention an isolated nucleic acidencoding an antibody, antigen binding fragment, or polypeptide asdescribed herein is provided. The nucleic acid may have a sequence ofone of SEQ ID NOs 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,or 88 (FIG. 4A-N), or a coding sequence which is degenerate as a resultof the genetic code, or may have a nucleotide sequence having at least70% identity thereto, optionally one of 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In one aspect of the present invention there is provided a vectorcomprising a nucleic acid described herein. In another aspect of thepresent invention, there is provided a host cell comprising the vector.For example, the host cell may be eukaryotic, or mammalian, e.g. ChineseHamster Ovary (CHO), or human or may be a prokaryotic cell, e.g. E.coli. In one aspect of the present invention a method for making anantibody, or antigen binding fragment or polypeptide as described hereinis provided, the method comprising culturing a host cell as describedherein under conditions suitable for the expression of a vector encodingthe antibody, or antigen binding fragment or polypeptide, and recoveringthe antibody, or antigen binding fragment or polypeptide.

In another aspect of the present invention an antibody, antigen bindingfragment or polypeptide is provided for use in therapy, or in a methodof medical treatment. In another aspect of the present invention anantibody, antigen binding fragment or polypeptide as described herein isprovided for use in the treatment of a T-cell dysfunctional disorder. Inanother aspect of the present invention, the use of an antibody, antigenbinding fragment or polypeptide as described herein in the manufactureof a medicament or pharmaceutical composition for use in the treatmentof a T-cell dysfunctional disorder is provided.

In another aspect of the present invention a method of enhancing T-cellfunction comprising administering an antibody, antigen binding fragmentor polypeptide as described herein to a dysfunctional T-cell isprovided. The method may be performed in vitro or in vivo.

In another aspect of the present invention a method of treating a T-celldysfunctional disorder is provided, the method comprising administeringan antibody, antigen binding fragment or polypeptide as described hereinto a patient suffering from a T-cell dysfunctional disorder.

In another aspect of the present invention a method of modulating animmune response in a subject is provided, the method comprisingadministering to the subject an antibody, antigen binding fragment orpolypeptide as described herein such that the immune response in thesubject is modulated.

In another aspect of the present invention a method of inhibiting growthof tumor cells in a subject is provided, the method comprisingadministering to the subject a therapeutically effective amount of anantibody, antigen binding fragment or polypeptide as described herein.

In another aspect of the present invention a method is provided, themethod comprising contacting a sample containing, or suspected tocontain, PD-1 with an antibody or antigen binding fragment, as describedherein, and detecting the formation of a complex of antibody, or antigenbinding fragment, and PD-1.

In another aspect of the present invention a method of diagnosing adisease or condition in a subject is provided, the method comprisingcontacting, in vitro, a sample from the subject with an antibody, orantigen binding fragment, as described herein, and detecting theformation of a complex of antibody, or antigen binding fragment, andPD-1.

In a further aspect of the present invention a method of selecting orstratifying a subject for treatment with PD-1 targeted agents isprovided, the method comprising contacting, in vitro, a sample from thesubject with an antibody, or antigen binding fragment, according to thepresent invention and detecting the formation of a complex of antibody,or antigen binding fragment, and PD-1.

In a further aspect of the present invention the use of an antibody, orantigen binding fragment, as described herein, for the detection of PD-1in vitro is provided. In another aspect of the present invention the useof an antibody, or antigen binding fragment, as described herein, as anin vitro diagnostic agent is provided.

In a further aspect of the present invention a method for expanding apopulation of T cells is provided, wherein T cells are contacted invitro or ex vivo with an antibody, antigen binding fragment orpolypeptide according to the present invention.

In a further aspect of the present invention a method of treatment of asubject having a T-cell dysfunctional disorder is provided, the methodcomprising culturing T cells obtained from a blood sample from a subjectin the presence of an antibody, antigen binding fragment or polypeptideaccording to the present invention so as to expand the T cellpopulation, collecting expanded T cells, and administering the expandedT cells to a subject in need of treatment.

In methods of the present invention the antibody, antigen bindingfragment or polypeptide may be provided as a composition as describedherein.

In some embodiments the antibody may be antibody clone A3, A10, B6, C4,D4, E1, F2, G1, G2, G10, H4, or H9 as described herein.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIGS. 1A-F. Light chain variable domain sequences for anti-PD-1 antibodyclones A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4, H9 (human IgG4).CDRs are underlined and shown separately.

FIGS. 2A-F. Heavy chain variable domain sequences for anti-PD-1 antibodyA3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4, H9 (human IgG4). CDRs areunderlined and shown separately.

FIGS. 3A-C. Table showing light chain and heavy chain CDR sequences foranti-PD-1 antibody clones A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4,H9.

FIGS. 4A-N. Nucleotide and encoded amino acid sequences of heavy andlight chain variable domain sequences for anti-PD-1 antibody clones A3,A10, optimised A10, B6, optimised B6, C4, optimised C4, D4, E1, F2, G1,G2, G10, H4, optimised H4 and H9 (human IgG4).

FIG. 5. Table showing binding affinity (K_(D), nM) of clones A3, A10,B6, C4, D4, E1, F2, G1, G2, G10, H4 and H9, and control antibodiesnivolumab and lambrolizumab for human PD-1.

FIG. 6. Chart showing binding of anti-human PD-1 antibodies A3, A10,nivolumab and lambrolizumab to activated T cells.

FIG. 7. Chart showing binding of anti-human PD-1 antibodies A10, B6, C4,D4, E1, F2, G1, G2, G10, H4 and H9, nivolumab and lambrolizumab toactivated T cells.

FIG. 8. Chart showing reactivity of clones B6, C4, D4, E1, F2, G1, G2,G10, H4 and H9 with human PD-1, rhesus PD-1 and human CTLA-4.

FIG. 9. Chart showing allogeneic CD4⁺ T cell proliferation of exhaustedT cells in response to antibodies A3, A10, nivolumab, lambrolizumab.

FIG. 10. Chart showing allogeneic CD4⁺ T cell proliferation of exhaustedT cells in response to antibodies A10, B6, C4, D4, E1, F2, G1, G2, G10,H4, H9, nivolumab, lambrolizumab.

FIG. 11. Chart showing IFNγ secretion of exhausted T cells in responseto antibodies A3, A10, nivolumab, lambrolizumab.

FIG. 12. Chart showing IFNγ secretion of exhausted T cells in responseto antibodies A10, B6, C4, D4, E1, F2, G1, G2, G10, H4, H9, nivolumab,lambrolizumab.

FIG. 13. Chart showing specificity of binding of antibodies A3, A10, B6,C4, D4, E1, F2, G1, G2, G10, H4, H9, for human and rhesus-PD-1 overother human (h) or murine (m) CD28 family members compared withnivolumab, and lambrolizumab, as determined by ELISA.

FIG. 14. Chart showing allogeneic CD4⁺ T cell proliferation of exhaustedT cells in response to antibodies A10 codon optimised, B6 codonoptimised, C4 codon optimised, H4 codon optimised, nivolumab,lambrolizumab.

FIG. 15. Chart showing IFNγ secretion of exhausted T cells in responseto antibodies A10 codon optimised, B6 codon optimised, C4 codonoptimised, H4 codon optimised, nivolumab, lambrolizumab.

FIG. 16. Chart showing the expression of exhaustion markers PD-1, PD-L1,TIM-3 and LAG-3 by lung tumour infiltrating lymphocytes. Approximately ⅔of CD4+ and CD8+ lymphocytes in the tumour express PD-1.

FIGS. 17A-B. Charts showing the secretion of IFN-γ by tumourinfiltrating lymphocytes (FIG. 17A) after 7 days of culture in thepresence or absence of anti-PD-1 antibodies (direct culture of tumourdissociated tissues), (FIG. 17B) after a mixed lymphocyte reaction inthe presence or absence of anti-PD-1 antibodies.

FIG. 18. Chart showing the secreted IFN-γ after culture of PBMCs withInfluenza virus infected dendritic cells in the presence or absence ofanti-PD-1 antibodies.

FIG. 19. Chart showing the expression of exhaustion markers PD-1, PD-L1,TIM-3, LAG-3 and CTLA4 by lung tumour infiltrating lymphocytes.Approximately ⅔ of CD4+ and CD8+ lymphocytes in the tumour express PD-1.

FIGS. 20A-C. Charts showing IFN-γ secretion by tumour infiltratinglymphocytes after a mixed lymphocyte reaction in the presence or absenceof anti-PD-1 antibodies, for three patients. (FIG. 20A) Patient #1,(FIG. 20B) Patient #2, (FIG. 20C) Patient #3. Shown are mean±SD fromduplicates or triplicates.

FIGS. 21A-B. Charts showing the expression of exhaustion markers PD-1,PD-L1, TIM-3, LAG-3 and CTLA4 by (FIG. 21A) renal tumour infiltratinglymphocytes and (FIG. 21B) blood circulating lymphocytes from renalcarcinoma patients. Approximately ⅔ of CD4+ and CD8+ lymphocytes in thetumour express PD-1, while most of PBMC lymphocytes do not express PD-1.

FIGS. 22A-C. Charts showing IFN-γ secretion by tumour infiltratinglymphocytes after a mixed lymphocyte reaction in the presence or absenceof anti-PD-1 antibodies, for three patients. (FIG. 22A) Patient #1,(FIG. 22B) Patient #2, (FIG. 22C) Patient #3. Shown are mean±SD fromduplicates or triplicates.

FIGS. 23A-B. Charts showing the expression of exhaustion markers PD-1,PD-L1, TIM-3, LAG-3 and CTLA4 by (FIG. 23A) bladder tumour infiltratinglymphocytes and (FIG. 23B) blood circulating lymphocytes from bladdercarcinoma patients. A majority of tumour infiltrating lymphocytesexpress PD-1, while only a minority of PBMC lymphocytes express PD-1.

FIG. 24. Chart showing IFN-γ secretion by tumour infiltratinglymphocytes after a mixed lymphocyte reaction in the presence or absenceof anti-PD-1 antibodies, for one patient. Shown are mean±SD fromduplicates or triplicates.

DESCRIPTION Antibodies

Antibodies according to the present invention preferably bind to PD-1(the antigen), preferably human or rhesus PD-1, optionally with a K_(D)in the range 0.1 to 2 nM.

In any aspect of the present invention the antibody preferablyspecifically binds PD-1 (e.g. human or rhesus) over other members of theCD28 family (preferably from the same organism), such as one or more oreach of TIM-3 (HAVCR2), LAG3 (CD223), ICOS (CD278), CTLA4 (CD152), BTLA(CD272) or CD28.

Antibodies according to the present invention may be provided inisolated form.

Antibodies according to the present invention may exhibit least one ofthe following properties:

-   -   a) binds to human PD-1 with a K_(D) of 1 μM or less, preferably        one of ≦10 nM, ≦1 nM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦400        pM, ≦300 pM, ≦200 pM or ≦100 pM;    -   b) does not substantially bind to human TIM-3, LAG3, ICOS,        CTLA4, BTLA or CD28;    -   c) increases T-cell proliferation in an Mixed Lymphocyte        Reaction (MLR) assay (e.g. see Bromelow et al J. Immunol        Methods, 2001 Jan. 1; 247(1-2):1-8);    -   d) increases interferon-gamma production in an MLR assay; or    -   e) increases interleukin-2 (IL-2) secretion in an MLR assay.

In some embodiments, the antibodies may be capable of increasinginterferon-gamma production in an MLR assay in a dose-dependent manner.In some embodiments, the antibodies may be capable of increasinginterferon-gamma production in an MLR assay by lymphocytes expressingone or more markers of exhaustion, e.g. PD-1.

By “antibody” we include a fragment or derivative thereof, or asynthetic antibody or synthetic antibody fragment.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies arediscussed by Neuberger et al (1988, 8th International BiotechnologySymposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are useful in the methods of the inventionand are a homogenous population of antibodies specifically targeting asingle epitope on an antigen.

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen binding fragments of antibodies, such as Fab and Fab₂ fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (V_(H)) and variable light(V_(L)) domains of the antibody are involved in antigen recognition, afact first recognised by early protease digestion experiments. Furtherconfirmation was found by “humanisation” of rodent antibodies. Variabledomains of rodent origin may be fused to constant domains of humanorigin such that the resultant antibody retains the antigenicspecificity of the rodent parented antibody (Morrison et al (1984) Proc.Natl. Acad. Sd. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodieswhich bind to PD-1 may also be made using phage display technology as iswell known in the art.

The present application also provides an antibody or antigen bindingfragment which is capable of binding to PD-1, and which is a bispecificantibody or a bispecific antigen binding fragment. In some embodiments,the bispecific antibody or bispecific antigen binding fragment may beisolated.

In some embodiments, the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding fragment or a polypeptideaccording to the present invention. In some embodiments, the bispecificantibodies and bispecific antigen binding fragments comprise an antigenbinding domain capable of binding to PD-1, wherein the antigen bindingdomain which is capable of binding to PD-1 comprises or consists of anantigen binding fragment or a polypeptide according to the presentinvention.

In some embodiments the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding domain capable of bindingto PD-1, and an antigen binding domain capable of binding to anothertarget protein.

The antigen binding domain capable of binding to another target proteinmay be capable of binding to another protein other than PD-1. In someembodiments, the target protein is a cell surface receptor. In someembodiments, the target protein is a cell surface receptor expressed onthe cell surface of immune cells, e.g. T cells. In some embodiments, thetarget protein may be a member of the CD28 family. In some embodiments,the target protein may be a member of the CD28 family such as TIM-3(HAVCR2), LAG3 (CD223), ICOS (CD278), CTLA4 (CD152), BTLA (CD272) orCD28. In particular embodiments, the target protein may be CTLA4 orLAG3.

In some embodiments, the antigen binding domain for TIM-3 may comprisethe CDRs, light and heavy chain variable domains or other TIM-3 bindingfragment of e.g. anti-TIM-3 antibody clone F38-2E2 (BioLegend), clone2E2 (Merck Millipore), clone 6136E2, clone 024 (Sino Biological) clone344801 (R&D Systems), clone E-18, clone H-191 (Santa CruzBiotechnology), or clone 13A224 (United States Biological). In someembodiments, the antigen binding domain for LAG3 may comprise the CDRs,light and heavy chain variable domains or other LAG3 binding fragment ofe.g. anti-LAG3 antibody clone 17134 (Enzo Life Sciences), clone 333210(R&D Systems), or clone 14L676 (United States Biological). In someembodiments, the antigen binding domain for ICOS may comprise the CDRs,light and heavy chain variable domains or other ICOS binding fragment ofe.g. anti-ICOS antibody clone ISA-3 (eBioscience), clone SP98 (NovusBiologicals), clone 1G1, clone 3G4 (Abnova Corporation), clone 669222(R&D Systems), clone TQ09 (Creative Diagnostics), or clone C398.4A(BioLegend). In some embodiments, the antigen binding domain for CTLA4may comprise the CDRs, light and heavy chain variable domains or otherCTLA4 binding fragment of e.g. anti-CTLA4 antibody clone 2F1, clone 1F4(Abnova Corporation), clone 9H10 (EMD Millipore), clone BNU3 (GeneTex),clone 1E2, clone AS32 (LifeSpan BioSciences) clone A3.4H2.H12 (AcrisAntibodies), clone 060 (Sino Biological), clone BU5G3 (CreativeDiagnostics), clone MIH8 (MBL International), clone A3.6B10.G1, or cloneL3D10 (BioLegend). In some embodiments, the antigen binding domain forBTLA may comprise the CDRs, light and heavy chain variable domains orother BTLA binding fragment of e.g. anti-BTLA antibody clone 1B7, clone2G8, clone 4C5 (Abnova Corporation), clone 4B8 (antibodies-online),clone MIH26 (Thermo Scientific Pierce Antibodies), clone UMAB61 (OriGeneTechnologies), clone 330104 (R&D Systems), clone 1B4 (LifeSpanBioSciences), clone 440205, clone 5E7 (Creative Diagnostics). In someembodiments, the antigen binding domain for CD28 may comprise the CDRs,light and heavy chain variable domains or other CD28 binding fragment ofe.g. anti-CD28 antibody clone CD28.6 (eBioscience), clone CD28.2, cloneJJ319 (Novus Biologicals), clone 204.12, clone B-23, clone 10F3 (ThermoScientific Pierce Antibodies), clone 37407 (R&D Systems), clone 204-12(Abnova Corporation), clone 15E8 (EMD Millipore), clone 204-12, cloneYTH913.12 (AbD Serotec), clone B-T3 (Acris Antibodies), clone 9H6E2(Sino Biological), clone C28/77 (MyBioSource.com), clone KOLT-2 (ALPCO),clone 152-2E10 (Santa Cruz Biotechnology), or clone XPH-56 (CreativeDiagnostics).

An antigen binding domain of a bispecific antibody or bispecific antigenbinding fragment according to the present invention may be any domain ofa polypeptide which is capable of binding to an antigen. In someembodiments, an antigen binding domain comprises at least the threelight chain CDRs (i.e. LC-CDR1, LC-CDR2 and LC-CDR3) and three heavychain CDRs (i.e. HC-CDR1, HC-CDR2 and HC-CDR3) which together define theantigen binding region of an antibody or antigen binding fragment. Insome embodiments, an antigen binding domain may comprise the light chainvariable domain and heavy chain variable domain of an antibody orantigen binding fragment. In some embodiments, an antigen binding domainmay comprise the light chain polypeptide and heavy chain polypeptide ofan antibody or antigen binding fragment.

Bispecific antibodies and bispecific antigen binding fragments accordingto the invention may be provided in any suitable format, such as thoseformats described in Kontermann MAbs 2012, 4(2): 182-197, which ishereby incorporated by reference in its entirety. For example, abispecific antibody or bispecific antigen binding fragment may be abispecific antibody conjugate (e.g. an IgG2, F(ab′)₂ or CovX-Body), abispecific IgG or IgG-like molecule (e.g. an IgG, scFv₄-Ig, IgG-scFv,scFv-IgG, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb², or Tandemab common LC), anasymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG, kih IgGcommon LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED-body), asmall bispecific antibody molecule (e.g. a Diabody (Db), dsDb, DART,scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triple body, triplehead, Fab-scFv, or F(ab′)₂-scFv₂), a bispecific Fc and C_(H)3 fusionprotein (e.g. a taFv-Fc, Di-diabody, scDb-C_(H)3, scFv-Fc-scFv,HCAb-VHH, scFv-kih-Fc, or scFv-kih-C_(H)3), or a bispecific fusionprotein (e.g. a scFv₂-albumin, scDb-albumin, taFv-toxin, DNL-Fab₃,DNL-Fab₄-IgG, DNL-Fab₄-IgG-cytokine₂). See in particular FIGS. 2A-F ofKontermann MAbs 2012, 4(2): 182-19.

The skilled person is able to design and prepare bispecific antibodiesand bispecific antigen binding fragments according to the presentinvention.

Methods for producing bispecific antibodies include chemicallycrosslinking of antibodies or antibody fragments, e.g. with reducibledisulphide or non-reducible thioether bonds, for example as described inSegal and Bast, 2001. Production of Bispecific Antibodies. CurrentProtocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which is herebyincorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH— groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers.

Other methods for producing bispecific antibodies include fusingantibody-producing hybridomas e.g. with polyethylene glycol, to producea quadroma cell capable of secreting bispecific antibody, for example asdescribed in D. M. and Bast, B. J. 2001. Production of BispecificAntibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments accordingto the present invention can also be produced recombinantly, byexpression from e.g. a nucleic acid construct encoding polypeptides forthe antigen binding molecules, for example as described in AntibodyEngineering: Methods and Protocols, Second Edition (Humana Press, 2012),at Chapter 40: Production of Bispecific Antibodies: Diabodies and TandemscFv (Hornig and Färber-Schwarz), or French, How to make bispecificantibodies, Methods Mol. Med. 2000; 40:333-339, the entire contents ofboth of which are hereby incorporated by reference.

For example, a DNA construct encoding the light and heavy chain variabledomains for the two antigen binding domains (i.e. the light and heavychain variable domains for the antigen binding domain capable of bindingPD-1, and the light and heavy chain variable domains for the antigenbinding domain capable of binding to another target protein), andincluding sequences encoding a suitable linker or dimerization domainbetween the antigen binding domains can be prepared by molecular cloningtechniques. Recombinant bispecific antibody can thereafter be producedby expression (e.g. in vitro) of the construct in a suitable host cell(e.g. a mammalian host cell), and expressed recombinant bispecificantibody can then optionally be purified.

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al,J. Mol. Biol. 226:889-896 (1992).

Antibodies according to the present invention preferably exhibitspecific binding to PD-1. An antibody that specifically binds to atarget molecule preferably binds the target with greater affinity,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by ELISA, or by a radioimmunoassay (RIA). Alternatively,the binding specificity may be reflected in terms of binding affinitywhere the anti-PD-1 antibody of the present invention binds to PD-1 witha K_(D) that is at least 0.1 order of magnitude (i.e. 0.1×10^(n), wheren is an integer representing the order of magnitude) greater than theK_(D) of the antibody towards another target molecule, e.g. anothermember of the CD28 family. This may optionally be one of at least 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

Antibodies according to the present invention preferably have adissociation constant (K_(D)) of one of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM or≦100 pM. Binding affinity of an antibody for its target is oftendescribed in terms of its dissociation constant (K_(D)). Bindingaffinity can be measured by methods known in the art, such as by SurfacePlasmon Resonance, or by a radiolabeled antigen binding assay (RIA)performed with the Fab version of the antibody and antigen molecule.

Antibodies according to the present invention may be “antagonist”antibodies that inhibit or reduce a biological activity of the antigento which it binds. Blocking of PD-1 assists in the restoration of T-cellfunction by inhibiting the immune-inhibitory signalling pathway mediatedby PD-1.

In some aspects, the antibody is clone A3, or a variant of A3. A3comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 27) ASWDDVLYGSV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 41) DLGAGPYYYGKDH

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone A10, or a variant of A10. A10comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 28) ASWDDYYYGTI Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 42) DLGAGPYYYGKDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone B6, or a variant of B6. B6comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 29) ASWDDYLRGTV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 43) DYGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone C4, or a variant of C4. C4comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 30) SAWDDYLHGTV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 44) DLGAGPYYYGLDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone D4, or a variant of D4. D4comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 31) ASWDDYVRGTM Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 45) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone E1, or a variant of E1. E1comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 32) SSWDDFLRGTV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 46) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone F2, or a variant of F2. F2comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 33) SSWDDDARGTI Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 47) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone G1, or a variant of G1. G1comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 34) AAWDDVYYGTI Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 48) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition

In some aspects, the antibody is clone G2, or a variant of G2. G2comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 35) ASWDDSLYGTV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 49) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone G10, or a variant of G10. G10comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 36) AAWDDAYYGTI Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 50) DYGAGPYYYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone H4, or a variant of H4. H4comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 37) ASWDDVYRGTV Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 51) DLGSGYYLYGMDV

CDR sequences determined by Kabat definition.

In some aspects, the antibody is clone H9, or a variant of H9. H9comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO:26) SNNQRPS LC-CDR3: (SEQ ID NO: 38) SSWDDSLYGTI Heavy chain: HC-CDR1:(SEQ ID NO: 39) GFTFSSYGMH or (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO:40) VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 52) DLGAGPYYYGMDV

CDR sequences determined by Kabat definition.

Antibodies according to the present invention may comprise the CDRs ofone of A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4, or H9 or one of SEQID NOs 1 and 13, 2 and 14, 3 and 15, 4 and 16, 5 and 17, 6 and 18, 7 and19, 8 and 20, 9 and 21, 10 and 22, 11 and 23, or 12 and 24. In anantibody according to the present invention one or two or three or fourof the six CDR sequences may vary. A variant may have one or two aminoacid substitutions in one or two of the six CDR sequences.

Amino acid sequences of the V_(H) and V_(L) chains of ant-PD-1 clonesare shown in FIGS. 1A-F and 2A-F. The encoding nucleotide sequences areshown in FIGS. 4A-N.

The light and heavy chain CDRs may also be particularly useful inconjunction with a number of different framework regions. Accordingly,light and/or heavy chains having LC-CDR1-3 or HC-CDR1-3 may possess analternative framework region. Suitable framework regions are well knownin the art and are described for example in M. Lefranc & G. Lefranc(2001) “The Immunoglobulin FactsBook”, Academic Press, incorporatedherein by reference.

In this specification, antibodies may have V_(H) and/or VL chainscomprising an amino acid sequence that has a high percentage sequenceidentity to one or more of the V_(H) and/or V_(L) amino acid sequencesof SEQ ID Nos 1 and 13, 2 and 14, 3 and 15, 4 and 16, 5 and 17, 6 and18, 7 and 19, 8 and 20, 9 and 21, 10 and 22, 11 and 23, or 12 and 24respectively, or to one or the amino acid sequences shown in FIGS. 1A-Fand 2A-F.

For example, antibodies according to the present invention includeantibodies that bind PD-1 and have a V_(H) or V_(L) chain that comprisesan amino acid sequence having at least 70%, more preferably one of atleast 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%, sequence identity to the V_(H) or V_(L)chain amino acid sequence of one of SEQ ID NOs 1 to 24 or to one or theamino acid sequences shown in FIGS. 1A-F and 2A-F.

Antibodies according to the present invention may be detectably labelledor, at least, capable of detection. For example, the antibody may belabelled with a radioactive atom or a coloured molecule or a fluorescentmolecule or a molecule which can be readily detected in any other way.Suitable detectable molecules include fluorescent proteins, luciferase,enzyme substrates, and radiolabels. The binding moiety may be directlylabelled with a detectable label or it may be indirectly labelled. Forexample, the binding moiety may be an unlabelled antibody which can bedetected by another antibody which is itself labelled. Alternatively,the second antibody may have bound to it biotin and binding of labelledstreptavidin to the biotin is used to indirectly label the firstantibody.

Methods of Detection

Antibodies, or antigen binding fragments, described herein may be usedin methods that involve the binding of the antibody or antigen bindingfragment to PD-1. Such methods may involve detection of the boundcomplex of antibody, or antigen binding fragment, and PD-1. As such, inone embodiment a method is provided, the method comprising contacting asample containing, or suspected to contain, PD-1 with an antibody orantigen binding fragment as described herein and detecting the formationof a complex of antibody, or antigen binding fragment, and PD-1.

Suitable method formats are well known in the art, includingimmunoassays such as sandwich assays, e.g. ELISA. The method may involvelabelling the antibody, or antigen binding fragment, or PD-1, or both,with a detectable label, e.g. fluorescent, luminescent or radio-label.

Methods of this kind may provide the basis of a method of diagnosis of adisease or condition requiring detection and or quantitation of PD-1.Such methods may be performed in vitro on a patient sample, or followingprocessing of a patient sample. Once the sample is collected, thepatient is not required to be present for the in vitro method ofdiagnosis to be performed and therefore the method may be one which isnot practised on the human or animal body.

Such methods may involve determining the amount of PD-1 present in apatient sample. The method may further comprise comparing the determinedamount against a standard or reference value as part of the process ofreaching a diagnosis. Other diagnostic tests may be used in conjunctionwith those described here to enhance the accuracy of the diagnosis orprognosis or to confirm a result obtained by using the tests describedhere.

The level of PD-1 present in a patient sample may be indicative that apatient may respond to treatment with an anti-PD1 antibody. The presenceof a high level of PD-1 in a sample may be used to select a patient fortreatment with an anti-PD1 antibody. The antibodies of the presentinvention may therefore be used to select a patient for treatment withanti-PD-1 therapy.

Detection in a sample of PD-1 may be used for the purpose of diagnosisof a T-cell dysfunctional disorder or a cancerous condition in thepatient, diagnosis of a predisposition to a cancerous condition or forproviding a prognosis (prognosticating) of a cancerous condition. Thediagnosis or prognosis may relate to an existing (previously diagnosed)cancerous condition, which may be benign or malignant, may relate to asuspected cancerous condition or may relate to the screening forcancerous conditions in the patient (which may be previouslyundiagnosed).

In one embodiment the level of PD-1 expression on CD8+ T cells may bedetected in order to indicate the degree of T-cell exhaustion andseverity of the disease state.

A sample may be taken from any tissue or bodily fluid. The sample maycomprise or may be derived from: a quantity of blood; a quantity ofserum derived from the individual's blood which may comprise the fluidportion of the blood obtained after removal of the fibrin clot and bloodcells; a tissue sample or biopsy; or cells isolated from saidindividual.

Methods according to the present invention are preferably performed invitro. The term “in vitro” is intended to encompass experiments withcells in culture whereas the term “in vivo” is intended to encompassexperiments with intact multi-cellular organisms.

Therapeutic Applications

Antibodies, antigen binding fragments and polypeptides according to thepresent invention and compositions comprising such agents may beprovided for use in methods of medical treatment. Treatment may beprovided to subjects having a disease or condition in need of treatment.The disease or condition may be one of a T-cell dysfunctional disorder,including a T-cell dysfunctional disorder associated with a cancer, or acancer, or a T-cell dysfunctional disorder associated with an infection,or an infection.

A T-cell dysfunctional disorder may be a disease or condition in whichnormal T-cell function is impaired causing downregulation of thesubject's immune response to pathogenic antigens, e.g. generated byinfection by exogenous agents such as microorganisms, bacteria andviruses, or generated by the host in some disease states such as in someforms of cancer (e.g. in the form of tumor associated antigens).

The T-cell dysfunctional disorder may comprise T-cell exhaustion orT-cell anergy. T-cell exhaustion comprises a state in which CD8⁺ T-cellsfail to proliferate or exert T-cell effector functions such ascytotoxicity and cytokine (e.g. IFNγ) secretion in response to antigenstimulation. Exhausted T-cells may also be characterised by sustainedexpression of PD-1, where blockade of PD-1:PD-L1 interactions mayreverse the T-cell exhaustion and restore antigen-specific T cellresponses.

The T-cell dysfunctional disorder may be manifest as an infection, orinability to mount an effective immune response against an infection.The infection may be chronic, persistent, latent or slow, and may be theresult of bacterial, viral, fungal or parasitic infection. As such,treatment may be provided to patients having a bacterial, viral orfungal infection. Examples of bacterial infections include infectionwith Helicobacter pylori. Examples of viral infections include infectionwith HIV, hepatitis B or hepatitis C.

The T-cell dysfunctional disorder may be associated with a cancer, suchas tumor immune escape. Many human tumors express tumor-associatedantigens recognised by T cells and capable of inducing an immuneresponse. However, immune evasion is common and is believed to bemediated by a number of soluble factors, including PD-L1. As such,blocking the interaction of PD-1 and PD-L1 may inhibit this negativeimmunoregulatory signal to tumor cells and enhance tumor-specific CD8⁺T-cell immunity.

Cancers may also be treated where there is no indication of a T-celldysfunctional disorder such as T-cell exhaustion but the use of anantibody, antigen binding fragment or polypeptide according to thepresent invention allows the subject to suppress PD-1 signalling andmount an effective immune response with limited impairment, evasion orinduction of tumor immune escape. In such treatments, the antibody,antigen binding fragment or polypeptide may provide a treatment forcancer that involves prevention of the development of tumor immuneescape.

The treatment may be aimed at prevention of the T-cell dysfunctionaldisorder, e.g. prevention of infection or of the development orprogression of a cancer. As such, the antibodies, antigen bindingfragments and polypeptides may be used to formulate pharmaceuticalcompositions or medicaments and subjects may be prophylactically treatedagainst development of a disease state. This may take place before theonset of symptoms of the disease state, and/or may be given to subjectsconsidered to be at greater risk of infection or development of cancer.

Treatment may comprise co-therapy with a vaccine, e.g. T-cell vaccine,which may involve simultaneous, separate or sequential therapy, orcombined administration of vaccine and antibody, antigen bindingfragment or polypeptide in a single composition. In this context, theantibody, antigen binding fragment or polypeptide may be provided as anadjuvant to the vaccine. Limited proliferative potential of exhausted Tcells has been attributed as a main reason for failure of T-cellimmunotherapy and combination an agent capable of blocking or reversingT cell exhaustion is a potential strategy for improving the efficacy ofT-cell immunotherapy (Barber et al., Nature Vol 439, No. 9 p 682-687February 2006).

Administration of an antibody, antigen binding fragment or polypeptideis preferably in a “therapeutically effective amount”, this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease being treated. Prescription oftreatment, e.g. decisions on dosage etc., is within the responsibilityof general practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Formulating Pharmaceutically Useful Compositions and Medicaments

Antibodies, antigen binding fragments and polypeptides according to thepresent invention may be formulated as pharmaceutical compositions forclinical use and may comprise a pharmaceutically acceptable carrier,diluent, excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating anantibody, antigen binding fragment or polypeptide as described herein;and/or mixing an isolated antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a medicament or pharmaceuticalcomposition for use in the treatment of a T-cell dysfunctional disorder,the method comprising formulating a pharmaceutical composition ormedicament by mixing an antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

Infection

An infection may be any infection or infectious disease, e.g. bacterial,viral, fungal, or parasitic infection. In some embodiments it may beparticularly desirable to treat chronic/persistent infections, e.g.where such infections are associated with T cell dysfunction or T cellexhaustion.

It is well established that T cell exhaustion is a state of T celldysfunction that arises during many chronic infections (including viral,bacterial and parasitic), as well as in cancer (Wherry Nature ImmunologyVol. 12, No. 6, p 492-499, June 2011).

An infection or infectious disease may be one in which PD-1 isupregulated (e.g. as reported by Radziewicz H, et al., J Virol. 2007;81(6):2545-2553 and Golden-Mason L et al., J Virol. 2007;81(17):9249-9258).

Examples of bacterial infections that may be treated include infectionby Bacillus spp., Bordetella pertussis, Clostridium spp.,Corynebacterium spp., Vibrio chloerae, Staphylococcus spp.,Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina,Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g.Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, thebacterial infection may be sepsis or tuberculosis.

Yao et al (PD-1 on dendritic cells impedes innate immunity againstbacterial infection. Blood 113(23):5811-5818 Jun. 4, 2009) establishedPD-1 in the negative regulation of DC function during innate immuneresponse to infection by Listeria monocytogenes. Brahmamdam et al(Delayed administration of anti-PD-1 antibody reverses immunedysfunction and improves survival during sepsis. Journal of LeukocyteBiology vo. 88, no. 2 233-240, August 2010) reported that anti-PD-1antibody administered 24 h after sepsis prevented sepsis-induceddepletion of lymphocytes and DCs, increased Bcl-xL, blocked apoptosisand improved survival. Tim3:Galectin-9 interactions have been reportedto mediate T cell exhaustion and mediate the innate and adaptive immuneresponse to infection by Mycobacterium tuberculosis (Jayaraman et al.,The Journal of Immunology 2012, 188, 70.6).

Examples of viral infections that may be treated include infection byinfluenza virus, measles virus, hepatitis B virus (HBV), hepatitis Cvirus (HCV), human immunodeficiency virus (HIV), lymphocyticchoriomeningitis virus (LCMV), Herpes simplex virus and human papillomavirus.

Chronic viral infections, such as those caused by HCV, HBV, and HIVcommonly involve mechanisms to evade immune clearance. Expression ofPD-1 and TIM-3 have been identified as correlating with defective T cellresponses to hepatitis C virus (HCV) (McMahan et al., The Journal ofClinical Investigation Vol. 120, No. 12 p 4546-4557, December 2010). InHCV, McMahan et al (supra) found that the level of dual TIM-3 and PD-1expression on HCV-specific CTLs predated the development of viralpersistence, providing prognostic information. Barber et al. (Nature Vol439, No. 9 p 682-687 February 2006) reported that PD-1 is upregulatedduring chronic viral infection. In mice infected with LCMV they reportedthat blockade of the PD-1/PD-L1 inhibitory pathway had a beneficialeffect on CD8 T cells, restoring their ability to undergo proliferation,secrete cytokines, kill infected cells and decrease viral load. PD-1 isalso upregulated in HIV infection (Said et al., Nature Medicine Vol. 16,No. 4 p 452-460 April 2010). Blocking interaction between PD-1 and PD-L1contributed to viral clearance and improved T cell function in animalmodels of chronic viral infection (Said et al., supra).

Examples of fungal infections that may be treated include infection byAlternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungalinfection may be fungal sepsis or histoplasmosis.

Chang et al (Blockade of the negative co-stimulatory molecules PD-1 andCTLA-4 improves survival in primary and secondary fungal sepsis.Critical Care 2013, 17:R85) reported that anti-PD1 antibodies werehighly effective at improving survival in primary and secondary fungalsepsis. Lázár-Molnár et al (The PD-1/PD-L costimulatory pathwaycritically affects host resistance to the pathogenic fungus Histoplasmacapsulatum PNAS vol. 105, no. 7, p 2658-2663, 19 Feb. 2008) reportedthat anti-PD-1 antibody significantly increased survival of miceinfected with Histoplasma capsulatum. As such, the importance of T cellexhaustion in mediating fungal infection is well established. Examplesof parasitic infections that may be treated include infection byPlasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli,Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi).The parasitic infection may be a disease such as malaria, leishmaniasisand toxoplasmosis.

Infection of humans with Plasmodium falciparum has been shown to resultin higher expression of PD-1 and T cell exhaustion mice (Butler et al.,Nature Immunology Vol. 13, No. 12, p 188-195 February 2012). Blockade ofPD-L1 and LAG-3 using anti-PD-L1 and anti-LAG-3 monoclonal antibodies invivo contributed to the restoration of CD4⁺ T-cell function,amplification of the number of follicular helper T cells,germinal-center B cells and plasmablasts, enhanced protective antibodiesand rapidly cleared blood-stage malaria in mice. It was also shown toblock the development of chronic infection (Butler et al., supra).

Cancer

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentume, oral cavity, ovary, pancreas, parotid gland, peripheralnervous system, peritoneum, pleura, prostate, salivary gland, sigmoidcolon, skin, small intestine, soft tissues, spleen, stomach, testis,thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, whiteblood cells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma.

In some embodiments, the cancer is one or more of lung cancer, renalcancer and bladder cancer.

Adoptive T Cell Transfer Therapy

Adoptive T cell transfer therapy generally refers to a process in whichwhite blood cells are removed from a subject, typically by drawing ablood sample from which white blood cells are separated, expanded invitro or ex vivo and returned either to the same subject or to adifferent subject. The treatment is typically aimed at increasing theamount/concentration of an active form of the required T cell populationin the subject. Such treatment may be beneficial in subjectsexperiencing T cell exhaustion.

Antibodies capable of blocking the mechanism of T cell exhaustion, orreversing it, provide a means of enhancing T cell activity and promotingT cell expansion.

Accordingly, in a further aspect of the present invention a method isprovided for expanding a population of T cells, wherein T cells arecontacted in vitro or ex vivo with an antibody, antigen binding fragmentor polypeptide according to the present invention.

The method may optionally comprise one or more of the following steps:taking a blood sample from a subject; isolating T cells from the bloodsample; culturing the T cells in in vitro or ex vivo cell culture (wherethey may be contacted with the antibody, antigen binding fragment orpolypeptide), collecting an expanded population of T cells; mixing the Tcells with an adjuvant, diluent, or carrier; administering the expandedT cells to a subject.

Accordingly, in some aspects of the present invention a method oftreatment of a subject having a T-cell dysfunctional disorder isprovided, the method comprising obtaining a blood sample from a subjectin need of treatment, culturing T cells obtained from the blood samplein the presence of an antibody, antigen binding fragment or polypeptideaccording to the present invention so as to expand the T cellpopulation, collecting expanded T cells, and administering the expandedT cells to a subject in need of treatment.

The T cells may be obtained from a subject requiring treatment, and maybe isolated and/or purified. They may be a CD4⁺ and/or CD8⁺ T-cellpopulation. The T-cells may represent a population experiencing T cellexhaustion and may optionally have upregulated expression of PD-1.

During culture, T cells may be contacted with the antibody, antigenbinding fragment or polypeptide under conditions and for a period oftime suitable to allow expansion of the T cells to a desired number ofcells. After a suitable period of time the T cells may be harvested,optionally concentrated, and may be mixed with a suitable carrier,adjuvant or diluent and returned to the subject's body. A subject mayundergo one or more rounds of such therapy.

Methods of T cell expansion are well known in the art, such as thosedescribed in Kalamasz et al., J Immunother 2004 September-October;27(5):405-18; Montes et al., Clin Exp Immunol 2005 November;142(2):292-302; Wölfl and Greenburg Nature Protocols 9 p 950-966 27 Mar.2014; Trickett and Kwan Journal of Immunological Methods Vol. 275,Issues 1-2, 1 Apr. 2003, p 251-255; Butler et al PLoSONE 7(1) 12 Jan.2012.

Simultaneous or Sequential Administration

Compositions may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

In this specification an antibody, antigen binding fragment orpolypeptide of the present invention and an anti-infective agent orchemotherapeutic agent (therapeutic agent) may be administeredsimultaneously or sequentially.

In some embodiments, treatment with an antibody, antigen bindingfragment or polypeptide of the present invention may be accompanied bychemotherapy.

Simultaneous administration refers to administration of the antibody,antigen binding fragment or polypeptide and therapeutic agent together,for example as a pharmaceutical composition containing both agents(combined preparation), or immediately after each other and optionallyvia the same route of administration, e.g. to the same artery, vein orother blood vessel.

Sequential administration refers to administration of one of theantibody, antigen binding fragment or polypeptide or therapeutic agentfollowed after a given time interval by separate administration of theother agent. It is not required that the two agents are administered bythe same route, although this is the case in some embodiments. The timeinterval may be any time interval.

Anti-Infective Agents

In treating infection, an antibody, antigen binding fragment orpolypeptide of the present invention may be administered in combinationwith an anti-infective agent, as described above. The anti-infectiveagent may be an agent known to have action against the microorganism orvirus responsible for the infection.

Suitable anti-infective agents include antibiotics (such as penicillins,cephalosporins, rifamycins, lipiarmycins, quinolones, sulfonamides,macrolides, lincosamides, tetracyclines, cyclic lipopeptides,glycylcyclines, oxazolidinones, and lipiarmycins), anti-viral agents(such as reverse transcriptase inhibitors, integrase inhibitors,transcription factor inhibitors, antisense and siRNA agents and proteaseinhibitors), anti-fungal agents (such as polyenes, imidiazoles,triazoles, thiazoles, allylamines, and echinocandins) and anti-parasiticagents (such as antinematode agents, anticestode agents, antitrematodeagents, antiamoebic agents and antiprotozoal agents).

Chemotherapy

Chemotherapy refers to treatment of a cancer with a drug or withionising radiation (e.g. radiotherapy using X-rays or γ-rays). Inpreferred embodiments chemotherapy refers to treatment with a drug. Thedrug may be a chemical entity, e.g. small molecule pharmaceutical,antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor),or a biological agent, e.g. antibody, antibody fragment, nucleic acid orpeptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, orprotein. The drug may be formulated as a pharmaceutical composition ormedicament. The formulation may comprise one or more drugs (e.g. one ormore active agents) together with one or more pharmaceuticallyacceptable diluents, excipients or carriers.

A treatment may involve administration of more than one drug. A drug maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. For example, the chemotherapy may be a co-therapy involvingadministration of two drugs, one or more of which may be intended totreat the cancer.

The chemotherapy may be administered by one or more routes ofadministration, e.g. parenteral, intravenous injection, oral,subcutaneous, intradermal or intratumoral.

The chemotherapy may be administered according to a treatment regime.The treatment regime may be a pre-determined timetable, plan, scheme orschedule of chemotherapy administration which may be prepared by aphysician or medical practitioner and may be tailored to suit thepatient requiring treatment.

The treatment regime may indicate one or more of: the type ofchemotherapy to administer to the patient; the dose of each drug orradiation; the time interval between administrations; the length of eachtreatment; the number and nature of any treatment holidays, if any etc.For a co-therapy a single treatment regime may be provided whichindicates how each drug is to be administered.

Chemotherapeutic drugs and biologics may be selected from:

-   -   alkylating agents such as cisplatin, carboplatin,        mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide;    -   purine or pyrimidine anti-metabolites such as azathiopurine or        mercaptopurine;    -   alkaloids and terpenoids, such as vinca alkaloids (e.g.        vincristine, vinblastine, vinorelbine, vindesine),        podophyllotoxin, etoposide, teniposide, taxanes such as        paclitaxel (Taxol™), docetaxel;    -   topoisomerase inhibitors such as the type I topoisomerase        inhibitors camptothecins irinotecan and topotecan, or the type        II topoisomerase inhibitors amsacrine, etoposide, etoposide        phosphate, teniposide;    -   antitumor antibiotics (e.g. anthracyline antibiotics) such as        dactinomycin, doxorubicin (Adriamycin™), epirubicin, bleomycin,        rapamycin;    -   antibody based agents, such as anti-TIM-3 antibodies,        anti-CTLA-4, anti-LAG-3, anti-4-1 BB, anti-GITR, anti-CD27,        anti-BLTA, anti-OX40, anti-VEGF, anti-TNFα, anti-IL-2,        antiGpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV,        anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies,        monoclonal antibodies or antibody fragments, examples include:        cetuximab, panitumumab, infliximab, basiliximab, bevacizumab        (Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab,        rituximab (Mabthera®), palivizumab, trastuzumab, etanercept,        adalimumab, nimotuzumab    -   EGFR inhibitors such as erlotinib, cetuximab and gefitinib    -   anti-angiogenic agents such as bevacizumab (Avastin®)    -   cancer vaccines such as Sipuleucel-T (Provenge®)

In one embodiment the chemotherapeutic agent is an anti-TIM-3 antibody,anti-CTLA-4, anti-LAGS, anti-41 BB, anti-GITR, anti-CD27, anti-BLTA,anti-OX40, anti-VEGF, anti-TNFα, anti-IL2, anti-GpIIb/IIIa, anti-CD-52,anti-CD20, anti-RSV, anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFRor other antibody. In some embodiments, the chemotherapeutic agent is animmune checkpoint inhibitor or costimulation molecule.

Further chemotherapeutic drugs may be selected from: 13-cis-RetinoicAcid, 2-Chlorodeoxyadenosine, 5-Azacitidine 5-Fluorouracil,6-Mercaptopurine, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-DAdriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin,Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®,Arsenic Trioxide, Asparaginase, ATRA Avastin®, Azacitidine, BCG, BCNU,Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU,Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, CalciumLeucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™,Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU,Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®,Cytarabine Cytosar-U®, Cytoxan®, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate, Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD,DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™, DTIC, DTIC-Dome®, Duralone®, Eligard™, Ellence™, Eloxatin™,Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, ErwiniaL-asparaginase, Estramustine, Ethyol Etopophos®, Etoposide, EtoposidePhosphate, Eulexin®, Everolimus, Evista®, Exemestane, Faslodex®,Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluoxymesterone, Flutamide, Folinic Acid, FUDR®,Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gleevec™,Gliadel® Wafer, Goserelin, Granulocyte-Colony Stimulating Factor,Granulocyte Macrophage Colony Stimulating Factor, Herceptin®, Hexadrol,Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, HydrocortAcetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate,Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea,Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®,IFN-alpha, Ifosfamide, IL-11, IL-2, Imatinib mesylate, ImidazoleCarboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase, Lanacort®,Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin,Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, LiposomalAra-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, LupronDepot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine,Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin® Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®.

Routes of Administration

Antibodies, antigen binding fragments, polypeptides and othertherapeutic agents, medicaments and pharmaceutical compositionsaccording to aspects of the present invention may be formulated foradministration by a number of routes, including but not limited to,parenteral, intravenous, intra-arterial, intramuscular, subcutaneous,intradermal, intratumoral and oral. Antibodies, antigen bindingfragments, polypeptides and other therapeutic agents, may be formulatedin fluid or solid form. Fluid formulations may be formulated foradministration by injection to a selected region of the human or animalbody.

Dosage Regime

Multiple doses of the antibody, antigen binding fragment or polypeptidemay be provided. One or more, or each, of the doses may be accompaniedby simultaneous or sequential administration of another therapeuticagent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

Kits

In some aspects of the present invention a kit of parts is provided. Insome embodiments the kit may have at least one container having apredetermined quantity of the antibody, antigen binding fragment orpolypeptide. The kit may provide the antibody, antigen binding fragmentor polypeptide in the form of a medicament or pharmaceuticalcomposition, and may be provided together with instructions foradministration to a patient in order to treat a specified disease orcondition. The antibody, antigen binding fragment or polypeptide may beformulated so as to be suitable for injection or infusion to a tumor orto the blood.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g.anti-infective agent or chemotherapy agent). In such embodiments, thekit may also comprise a second medicament or pharmaceutical compositionsuch that the two medicaments or pharmaceutical compositions may beadministered simultaneously or separately such that they provide acombined treatment for the specific disease or condition. Thetherapeutic agent may also be formulated so as to be suitable forinjection or infusion to a tumor or to the blood.

Subjects

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient. A subject may have beendiagnosed with a disease or condition requiring treatment, or besuspected of having such a disease or condition.

Protein Expression

Molecular biology techniques suitable for the producing polypeptidesaccording to the invention in cells are well known in the art, such asthose set out in Sambrook et al., Molecular Cloning: A LaboratoryManual, New York: Cold Spring Harbor Press, 1989

The polypeptide may be expressed from a nucleotide sequence. Thenucleotide sequence may be contained in a vector present in a cell, ormay be incorporated into the genome of the cell.

A “vector” as used herein is an oligonucleotide molecule (DNA or RNA)used as a vehicle to transfer exogenous genetic material into a cell.The vector may be an expression vector for expression of the geneticmaterial in the cell. Such vectors may include a promoter sequenceoperably linked to the nucleotide sequence encoding the gene sequence tobe expressed. A vector may also include a termination codon andexpression enhancers. Any suitable vectors, promoters, enhancers andtermination codons known in the art may be used to express polypeptidesfrom a vector according to the invention. Suitable vectors includeplasmids, binary vectors, viral vectors and artificial chromosomes (e.g.yeast artificial chromosomes).

In this specification the term “operably linked” may include thesituation where a selected nucleotide sequence and regulatory nucleotidesequence (e.g. promoter and/or enhancer) are covalently linked in such away as to place the expression of the nucleotide sequence under theinfluence or control of the regulatory sequence (thereby forming anexpression cassette). Thus a regulatory sequence is operably linked tothe selected nucleotide sequence if the regulatory sequence is capableof effecting transcription of the nucleotide sequence. Whereappropriate, the resulting transcript may then be translated into adesired protein or polypeptide.

Any cell suitable for the expression of polypeptides may be used forproducing peptides according to the invention. The cell may be aprokaryote or eukaryote. Suitable prokaryotic cells include E. coli.Examples of eukaryotic cells include a yeast cell, a plant cell, insectcell or a mammalian cell. In some cases the cell is not a prokaryoticcell because some prokaryotic cells do not allow for the samepost-translational modifications as eukaryotes. In addition, very highexpression levels are possible in eukaryotes and proteins can be easierto purify from eukaryotes using appropriate tags. Specific plasmids mayalso be utilised which enhance secretion of the protein into the media.

Methods of producing a polypeptide of interest may involve culture orfermentation of a cell modified to express the polypeptide. The cultureor fermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted polypeptide.Culture, fermentation and separation techniques are well known to thoseof skill in the art.

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culture of cells that express the polypeptide of interest,that polypeptide is preferably isolated. Any suitable method forseparating polypeptides/proteins from cell culture known in the art maybe used. In order to isolate a polypeptide/protein of interest from aculture, it may be necessary to first separate the cultured cells frommedia containing the polypeptide/protein of interest. If thepolypeptide/protein of interest is secreted from the cells, the cellsmay be separated from the culture media that contains the secretedpolypeptide/protein by centrifugation. If the polypeptide/protein ofinterest collects within the cell, it will be necessary to disrupt thecells prior to centrifugation, for example using sonification, rapidfreeze-thaw or osmotic lysis. Centrifugation will produce a pelletcontaining the cultured cells, or cell debris of the cultured cells, anda supernatant containing culture medium and the polypeptide/protein ofinterest.

It may then be desirable to isolate the polypeptide/protein of interestfrom the supernatant or culture medium, which may contain other proteinand non-protein components. A common approach to separatingpolypeptide/protein components from a supernatant or culture medium isby precipitation. Polypeptides/proteins of different solubility areprecipitated at different concentrations of precipitating agent such asammonium sulfate. For example, at low concentrations of precipitatingagent, water soluble proteins are extracted. Thus, by adding increasingconcentrations of precipitating agent, proteins of different solubilitymay be distinguished. Dialysis may be subsequently used to removeammonium sulfate from the separated proteins.

Other methods for distinguishing different polypeptides/proteins areknown in the art, for example ion exchange chromatography and sizechromatography. These may be used as an alternative to precipitation, ormay be performed subsequently to precipitation.

Once the polypeptide/protein of interest has been isolated from cultureit may be necessary to concentrate the protein. A number of methods forconcentrating a protein of interest are known in the art, such asultrafiltration or lyophilisation.

Sequence Identity

Alignment for purposes of determining percent amino acid or nucleotidesequence identity can be achieved in various ways known to a person ofskill in the art, for instance, using publicly available computersoftware such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software.When using such software, the default parameters, e.g. for gap penaltyand extension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNAGAPDIST=4.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

Examples

The inventors described in the following Examples the identification ofnucleotide and amino-acid sequences of isolated antibodies, or theantigen-binding portions thereof, that specifically bind human andrhesus PD-1, block the PD-1 pathway and restore exhausted T cellactivity.

Isolation of Anti-Human PD-1 Antibodies

Anti-PD-1 antibodies were isolated from a human antibody phage displaylibrary via in vitro selection in a 4-round bio-panning process.

Streptavidin-magnetic beads were coated with biotinylated human PD-1 andused to fish-out anti-PD-1-specific phages using magnetic sorting. Somesteps to remove of potential anti-biotin antibodies were added in theselection process.

Specific Fab antibodies were originally identified by ELISA withhuman-PD-1 as the antigen. A first clonality screening was performed byDNA fingerprinting; clonality was then confirmed by sequencing.

Affinity Maturation

Selected antibodies went through affinity maturation by CDR engineering.Selection of specific anti-PD-1 antibodies was conducted by phagedisplay. More than 80 affinity-matured clones with ˜100-fold improvedaffinity were generated. To select clones, thermostability assays wereconducted. Antibodies were heated and their stability monitored based ontheir ability to bind human PD-1 in ELISA. Clones still stable at 71° C.were kept for further analyses, their affinity for human PD-1 wasmeasured using a ProteOn bioanalyser (Biorad). Briefly, human-PD-1coupled to Fc was immobilised on a sensor chip and a flow of antibodywas applied; association and dissociation rates were measured and theaffinity (K_(D)) calculated. Twelve of the affinity-matured clones weredesignated A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4 and H9.

Affinity of the Isolated Anti-PD-1 Antibodies

Affinity of A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4 and H9 forhuman PD-1 was measured by Surface Plasmon Resonance and compared to theaffinity of nivolumab and lambrolizumab, two anti-PD-1 antibodies inlate clinical development stage (FIG. 5).

Briefly, human or mouse PD-1 coupled to human Fc was immobilised on asensor chip compatible with the Proteon XPR36 bioanalyser (Biorad).Crude Fab extracts (or the control antibodies) were then flown onto thechip and the association/dissociation of each candidate Fab was thenrecorded and analysed, the affinity (K_(D)) was calculated.

A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4 and H9 show affinity in therange 0.1 to 0.7 nM while nivolumab and lambrolizumab exhibit K_(D) of1.9 and 0.5 nM respectively (FIG. 5).

Characterisation of the Anti-Human PD-1 Antibodies: Binding to PD-1Expressing Cells

50,000 T cells isolated from a healthy donor were incubated in thepresence of 5,000 monocyte-derived DCs from another donor for 7 days at37° C. Antibodies, expressed as IgG4, were incubated with such activatedT cells for 30 minutes in PBS. After a wash in PBS, a fluorescentlabelled secondary antibody was added for 30 minutes and then washed inPBS. Cells were resuspended in FACS buffer and binding of the antibodyto the cells was monitored by flow cytometry (FIGS. 6 and 7).

Cross-Reactivity with Rhesus PD-1

Affinity-matured clone Fabs were tested in ELISA for recognition ofrhesus PD-1. Briefly, ELISA plates were coated with 350 ng/well of humanor rhesus PD-1 in carbonate buffer and then blocked with a solution ofcasein. After extensive washes in PBS Tween-20, antibody-containingsupernatants were transferred into the ELISA plates in the presence of7% milk in PBS. After 90 minutes at room temperature under agitation andextensive washes, a goat anti-human Fab antibody coupled to HRP wasadded. One hour later, plates were washed and TMB substrate added. Thereaction was stopped with 1M HCl and optical density measured at 450 nmwith a reference at 670 nm (FIG. 8).

In Vitro Functional Activity

Anti-PD-1 antibodies were tested in a functional assay measuring 2activity parameters of T cells: proliferation and secretion of IFN-γ.Briefly, T cells were isolated from a healthy donor and cultured for 7days in the presence of monocyte-derived dendritic cells from anotherdonor (50,000 Tcells/5,000 DCs). This continuous stimulation inducesexhaustion of the T cells. Antibodies were then added to the culturesfor 5 more days. After 4 days, supernatants were collected to measureIFN-γ by ELISA, and T cells were cultured for 4 days, with addition of 1μCi of tritiated thymidine for the last 18 hours. Cells were thenharvested and proliferation measured with a β-counter.

Clones A3, A10, B6, C4, D4, E1, F2, G1, G2, G10, H4 and H9 are able torestore proliferation of previously exhausted T cells and to restoretheir ability to secrete IFN-γ (FIGS. 9 to 12).

Specificity for PD-1

Anti-PD-1 antibodies were tested in an ELISA assay for their ability tobind to other members of the CD28 family.

Briefly, ELISA plates were coated with 350 ng/well of one of thefollowing antigens coupled to human Fc in carbonate buffer: human PD-1,human PD-L1, human TIM-3, human LAG-3, human ICOS, human CTLA4, humanBTLA, human CD28, mouse TIM-3, or rhesus PD-1. The plate was thenblocked with a solution of casein. After extensive washes in PBSTween-20, antibodies were added into the ELISA wells in the presence of7% milk in PBS. After 90 minutes at room temperature under agitation andextensive washes, a goat anti-human Fab antibody coupled to HRP wasadded. One hour later, plates were washed and TMB substrate added. Thereaction was stopped with 1M HCl and optical density measured at 450 nmwith a reference at 670 nm.

While nivolumab cross-binds to PD-L1, TIM-3, LAG-3, BTLA and CD28 (andlambrolizumab in a very weak manner too), 1D11 and 1G4 are specific toPD-1 only; showing only a very weak cross-recognition of other membersthan PD-1 (FIG. 13).

Production Yield

HEK-293.6E cells were transiently transfected to produce anti-PD-1antibodies in IgG4 format; production yield in this system was compared.

Antibody Production yield (mg/L) Lambrolizumab 2.1 A3 9.9 A10 32.1 E130.6 F2 32.6 G1 30.8 G2 28.8 H9 18.4 D4 26.9 B6 26.5 C4 27.4 H4 19.8 G1033.6

All novel antibody clones showed better production yield thanlambrolizumab.

Codon Optimisation

In order to increase gene expression efficiency, A10, B6, C4 and H4underwent codon optimisation.

Codon-optimised clones retain their ability to neutralise PD-1 andrestore T cell activity (T cell proliferation and IFN-γ secretion—FIGS.14 and 15).

Use of Anti-PD-1 Antibodies to Treat Tumours: Ex Vivo Activation ofTumour Infiltrating Lymphocytes

Lung tumour samples were obtained from the National Cancer CentreSingapore after approval by the proper IRB. Samples were dissociatedusing a human tumour dissociation kit and a tissue dissociator device.

To confirm expression of exhaustion markers at the surface of the tumourinfiltrating lymphocytes, isolated cell mixture was washed once andpassed through 70 μm filter to obtain single cell suspension. Cells werestained with antibodies against CD4, CD8, PD-1, PD-L1, TIM-3 and LAG-3;a live/dead marker was also used to exclude dead cells from theanalysis. Cells were analysed by flow cytometry. Results are shown inFIG. 16.

The tumour dissociated mixture was cultured with anti-PD-1codon-optimised antibodies A10, B6, C4 and H4 for 7 days prior tomeasurement of IFN-γ in the supernatant by ELISA. Nivolumab andlambrolizumab were used as positive controls, an isotype antibody as anegative control.

FIG. 17A presents the secretion of IFN-γ by tumour infiltratinglymphocytes after 7 days of culture in the presence or absence ofanti-PD-1 antibodies. Anti-PD-1 antibodies were able to re-activatelymphocytes to secrete IFN-γ in a dose-dependent manner.

Another fraction of the dissociated mixture was co-cultured withallogeneic dendritic cells (DCs) to initiate a mixed lymphocyte reaction(MLR). Cells were first cultured with DCs for 7 days without antibodiesand then restimulated with DCs for 7 days in the presence of anti-PD-1or control antibodies. After these 2 rounds, IFN-γ was assayed insupernatants by ELISA.

FIG. 17B presents the secretion of IFN-γ after the MLR in the presenceor absence of anti-PD-1 antibodies. Anti-PD-1 antibodies were able torestore the ability of lymphocytes located in the tumour site to secreteIFN-γ in a dose-dependent manner.

Use of Anti-PD-1 Antibodies to Treat Infections: Autologous Activationof T Cells in the Presence of Influenza

Blood was collected from Influenza-positive donors. Monocyte-derived DCswere infected with influenza virus A/PR/8/34 (H1N1). Infected DCs werethen mixed to PBMCs from the same donor for a first round of culture of5 days. Cells were then restimulated with Influenza-infected DCs andcultured for a second round of 5 days in the presence of anti-PD-1antibodies. After these 2 rounds, most of the cells in culture areInfluenza-specific T cells. At the end of the 2 rounds of culture, IFN-γwas assayed in supernatants by ELISA.

FIG. 18 presents the secreted IFN-γ after culture of PBMCs withInfluenza virus infected DCs in the presence or absence of anti-PD-1antibodies. Anti-PD-1 antibodies were able to restore the capacity oflymphocytes to secrete IFN-γ upon viral stimulation in a dose-dependentmanner.

Luna Cancer: Use of Anti-PD-1 Antibodies to Re-Activate Lung TumourInfiltrating Lymphocytes (Ex Vivo Data)

Lung tumour samples were obtained from the National Cancer CentreSingapore after approval by the proper IRB. Samples were dissociatedusing a human tumour dissociation kit and a tissue dissociator device.

To confirm expression of exhaustion markers at the surface of the tumourinfiltrating lymphocytes, isolated cell mixture was washed once andpassed through 70 μm filter to obtain single cell suspension. Cells werestained with antibodies against CD4, CD8, PD-1, PD-L1, Tim-3, LAG-3 andCTLA-4; a live/dead marker was also used to exclude dead cells from theanalysis. Cells were analysed by flow cytometry.

FIG. 19 shows the expression of PD-1, PD-L1, TIM-3, LAG-3 and CTLA-4 bytumour infiltrating lymphocytes from 3 different patients (shown aremean±SD from 3 independent experiments using cells from 3 differentdonors, all experiments done in triplicates). Approximately ⅔ of CD4+and CD8+ lymphocytes in the tumour express PD-1.

The tumour dissociated mixture was co-cultured with allogeneic dendriticcells (DC) to initiate a mixed lymphocyte reaction (MLR). Cells werefirst cultured for 7 days without antibodies and then for 7 days in thepresence of anti-PD-1 or control antibodies. After these 2 rounds, IFN-γwas assayed in supernatants by ELISA.

FIGS. 20A-20C present the secretion of IFN-γ after the MLR in thepresence or absence of anti-PD-1 antibodies. Shown are mean±SD fromtriplicates in 3 independent experiments (cells from 3 differentpatients). Anti-PD-1 antibodies were able to restore the ability oflymphocytes located in the tumour site to secrete IFN-γ in adose-dependent manner.

Renal Cell Carcinoma: Use of Anti-PD-1 Antibodies to Re-Activate KidneyTumour Infiltrating Lymphocytes (Ex Vivo Data)

Kidney tumour samples were obtained from the National Cancer CentreSingapore after approval by the proper IRB. Samples were dissociatedusing a human tumour dissociation kit and a tissue dissociator device.

To confirm expression of exhaustion markers at the surface of the tumourinfiltrating lymphocytes, isolated cell mixture was washed once andpassed through 70 μm filter to obtain single cell suspension. Cells werestained with antibodies against CD4, CD8, PD-1, PD-L1, Tim-3, LAG-3 andCTLA-4; a live/dead marker was also used to exclude dead cells from theanalysis. Cells were analysed by flow cytometry. As a comparison, samemarkers expression was assessed on PBMCs from the same patients.

FIGS. 21A and 21B show the expression of PD-1, PD-L1, TIM-3, LAG-3 andCTLA-4 by tumour infiltrating lymphocytes (FIG. 21A) and on bloodcirculating lymphocytes (FIG. 21B) from 3 different patients (shown aremean±SD from 3 independent experiments using cells from 3 differentdonors, all experiments done in triplicates). Approximately ⅔ of CD4+and CD8+ lymphocytes in the tumour express PD-1 while most of PBMClymphocytes do not express PD-1.

The tumour dissociated mixture was co-cultured with allogeneic dendriticcells (DC) to initiate a mixed lymphocyte reaction (MLR). Cells werefirst cultured for 7 days without antibodies and then for 7 days in thepresence of anti-PD-1 or control antibodies. After these 2 rounds, IFN-γwas assayed in supernatants by ELISA.

FIGS. 22A-22C present the secretion of IFN-γ after the MLR in thepresence or absence of anti-PD-1 antibodies. Shown are mean±SD fromduplicates or triplicates in 3 independent experiments (cells from 3different patients). Anti-PD-1 antibodies were able to restore theability of lymphocytes located in the tumour site to secrete IFN-γ in adose-dependent manner.

Bladder Cancer: Use of Anti-PD-1 Antibodies to Re-Activate BladderTumour Infiltrating Lymphocytes (Ex Vivo Data)

Bladder tumour samples were obtained from the National Cancer CentreSingapore after approval by the proper IRB. Samples were dissociatedusing a human tumour dissociation kit and a tissue dissociator device.

To confirm expression of exhaustion markers at the surface of the tumourinfiltrating lymphocytes, isolated cell mixture was washed once andpassed through 70 μm filter to obtain single cell suspension. Cells werestained with antibodies against CD4, CD8, PD-1, PD-L1, Tim-3, LAG-3 andCTLA-4; a live/dead marker was also used to exclude dead cells from theanalysis. Cells were analysed by flow cytometry.

FIGS. 23A and 23B show the expression of PD-1, PD-L1, TIM-3, LAG-3 andCTLA-4 by tumour infiltrating lymphocytes (FIG. 23A) and bloodcirculating lymphocytes (FIG. 23B) from 2 different patients (shown aremean±SD from 2 independent experiments using cells from 2 differentdonors, all experiments done in triplicates). A majority of tumourinfiltrating lymphocytes express PD-1, while only a minority of theirblood circulating counterparts do so.

The tumour dissociated mixture was co-cultured with allogeneic dendriticcells (DC) to initiate a mixed lymphocyte reaction (MLR). Cells werefirst cultured for 7 days without antibodies and then for 7 days in thepresence of anti-PD-1 or control antibodies. After these 2 rounds, IFN-γwas assayed in supernatants by ELISA.

FIG. 24 presents the secretion of IFN-γ after the MLR in the presence orabsence of anti-PD-1 antibodies. Shown are mean±SD from triplicates in 1experiment. Anti-PD-1 antibodies were able to restore the ability oflymphocytes located in the tumour site to secrete IFN-γ in adose-dependent manner.

1. A method of treating cancer, comprising administering an antibody orantigen binding fragment to a patient suffering from a cancer, whereinthe antibody or antigen binding fragment binds to PD-1, and comprises:at least one light chain variable region incorporating the followingCDRs: LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26)SNNQRPS LC-CDR3: (SEQ ID NO: 29) ASWDDYLRGTV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO: 40)VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 43) DYGAGPYYYGMDV.


2. The method of claim 1, wherein the antibody or antigen bindingfragment comprises a heavy chain variable region sequence having atleast 85% sequence identity to the amino acid sequence of SEQ ID NO:15,and a light chain variable region sequence having at least 85% sequenceidentity to the amino acid sequence of SEQ ID NO:3.
 3. The method ofclaim 1, wherein the antibody or antigen binding fragment comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:15, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:3.
 4. The method of claim 1, wherein the cancer isa cancer of a tissue selected from the group consisting of lung, kidney,bladder, liver, stomach, cervix, nasopharynx, oral cavity, oesophagus,larynx, salivary gland, tongue, tonsil, trachea, skin, blood, colon andbreast.
 5. The method of claim 1, wherein the cancer is selected fromthe group consisting of lung cancer, non-small cell lung cancer (NSCLC),renal cancer, renal cell carcinoma, bladder cancer, bladder carcinoma,liver cancer, hepatoma, stomach cancer, cervical cancer, nasopharyngealcancer, oral cavity cancer, oesophageal cancer, laryngeal cancer,salivary gland cancer, tongue cancer, tonsil cancer, tracheal cancer,skin cancer, melanoma, metastatic melanoma, haematologic cancer,lymphoma, Hodgkin's lymphoma, colon cancer, colon carcinoma and breastcancer.
 6. The method of claim 1, wherein the administration of theantibody or antigen binding fragment is intravenous.
 7. The method ofclaim 1, wherein the antibody comprises a human constant region selectedfrom IgG1, IgG2, IgG3 and IgG4.
 8. The method of claim 1, wherein theantigen binding fragment is a Fab fragment or scFv fragment.
 9. A methodof treating cancer, comprising administering an antibody or antigenbinding fragment to a patient suffering from a cancer, wherein theantibody or antigen binding fragment binds to PD-1, and comprises: aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:15, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:3.
 10. The method of claim 9, wherein the canceris a cancer of a tissue selected from the group consisting of lung,kidney, bladder, liver, stomach, cervix, nasopharynx, oral cavity,oesophagus, larynx, salivary gland, tongue, tonsil, trachea, skin,blood, colon and breast.
 11. The method of claim 9, wherein the canceris selected from the group consisting of lung cancer, non-small celllung cancer (NSCLC), renal cancer, renal cell carcinoma, bladder cancer,bladder carcinoma, liver cancer, hepatoma, stomach cancer, cervicalcancer, nasopharyngeal cancer, oral cavity cancer, oesophageal cancer,laryngeal cancer, salivary gland cancer, tongue cancer, tonsil cancer,tracheal cancer, skin cancer, melanoma, metastatic melanoma,haematologic cancer, lymphoma, Hodgkin's lymphoma, colon cancer, coloncarcinoma and breast cancer.
 12. The method of claim 9, wherein theadministration of the antibody or antigen binding fragment isintravenous.
 13. The method of claim 9, wherein the antibody comprises ahuman constant region selected from IgG1, IgG2, IgG3 and IgG4.
 14. Themethod of claim 9, wherein the antigen binding fragment is a Fabfragment or scFv fragment.
 15. A method of treating cancer in a subject,comprising: culturing T cells obtained from a blood sample from asubject having a cancer in the presence of an antibody or antigenbinding fragment which binds to PD-1 to expand a T cell population; andadministering the expanded T cell population to the subject; wherein theantibody or antigen binding fragment which binds to PD-1 comprises: atleast one light chain variable region incorporating the following CDRs:LC-CDR1: (SEQ ID NO: 25) SGSSSNIKFNSVN LC-CDR2: (SEQ ID NO: 26) SNNQRPSLC-CDR3: (SEQ ID NO: 29) ASWDDYLRGTV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 89) SYGMH HC-CDR2: (SEQ ID NO: 40)VISYDGSNKYYADSVKG HC-CDR3: (SEQ ID NO: 43) DYGAGPYYYGMDV.


16. The method of claim 15, wherein the antibody or antigen bindingfragment comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:15, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:3.
 17. The method ofclaim 15, wherein the cancer is a cancer of a tissue selected from thegroup consisting of lung, kidney, bladder, liver, stomach, cervix,nasopharynx, oral cavity, oesophagus, larynx, salivary gland, tongue,tonsil, trachea, skin, blood, colon and breast.
 18. The method of claim15, wherein the cancer is selected from the group consisting of lungcancer, non-small cell lung cancer (NSCLC), renal cancer, renal cellcarcinoma, bladder cancer, bladder carcinoma, liver cancer, hepatoma,stomach cancer, cervical cancer, nasopharyngeal cancer, oral cavitycancer, oesophageal cancer, laryngeal cancer, salivary gland cancer,tongue cancer, tonsil cancer, tracheal cancer, skin cancer, melanoma,metastatic melanoma, haematologic cancer, lymphoma, Hodgkin's lymphoma,colon cancer, colon carcinoma and breast cancer.
 19. The method of claim15, wherein the antibody comprises a human constant region selected fromIgG1, IgG2, IgG3 and IgG4.
 20. The method of claim 15, wherein theantigen binding fragment is a Fab fragment or scFv fragment.